Abstract
In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.
Highlights
Connexin 43 hemichannels participate in many cellular processes
Production and Purification of connexin 43 (Cx43) and Cx43-green fluorescent protein (GFP)—Mouse Cx43 with a hexahistidine tag at the C terminus for affinity purification was recombinantly expressed in P. pastoris
For visualization of Cx43 in artificial membranes, Cx43 was fused to green fluorescent protein with a single point mutation of S65T in the original GFP sequence to enhance GFP fluorescence emission
Summary
Connexin 43 hemichannels participate in many cellular processes. To elucidate their location and function within living cells, they were labeled with GFP. The analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. Processes such as development, differentiation, and maintenance of nutrients in tissues of vertebrates rely on a controlled exchange of intracellular solutes. A further hallmark of the broad functional spectrum of Cx43 is the rare disease oculodentodigital dysplasia, which causes the
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